In order to analyze the genetic regulation of T cell responses to NASE, a series of cloned lines were generated in BALB/c (H-2d) as well as (H-2b x H-2a)F1 T cells. Individual clones were restricted to recognizing NASE in the context of either A(alpha)A(beta) or E(alpha)E(beta) products. The antigen fine specificity of cloned NASE-specific T cells was also probed through the use of mutant NASE molecules and synthetic peptides.A consistent correlation was found between the fine specificity of a given clone and its MHC restriction specificity. A(alpha)(b)A(beta)(b) restricted clones were selectively responsive to peptide 91-110; E(alpha)(k)E(beta)(k) restricted clones were responsive to peptide 81- 100. The nature of the immunogenic peptides which are processed and presented to T cells was further evaluated using a panel of variant NASE peptides. It was observed that negative (inhibitory) interactions appear to occur between amino acids in peptides which interfere with T cell responses. Single amino acid changes, by eliminating such apparent negative interactions, result in the restoration of T cell stimulatory ability in these peptides. The T cell receptors (TCR) expressed by NASE specific clones have been analyzed to determine the relationship between antigenic fine specificity and TCR expression. Preferential expression of specific V-alpha and V- beta products was observed, e.g.expression of V-beta4 by six of seven independent clones specific for NASE 91-110 in association with A(alpha)(b)A(beta)(b), and expression of V-beta10 in five of nine independent clones specific for NASE 81-100 in association with E(alpha)(k)E(beta)(k). Conservation of receptor usage was also reflected in sequence analysis by PCR amplification of alpha and beta chain cDNA.